Preventative Cancer Vaccine-Elicited Human Anti-MUC1 Antibodies Have Multiple Effector Functions.

BACKGROUND/OBJECTIVES: Mucin-1 (MUC1) is a transmembrane glycoprotein that is overexpressed and hypoglycosylated in premalignant and malignant epithelial cells compared to normal cells, creating a target antigen for humoral and cellular immunity. Healthy individuals with a history of advanced colonic adenomas and at high risk for colon cancer were enrolled in a clinical trial to evaluate the feasibility of using a MUC1 peptide vaccine to prevent colon cancer. Anti-MUC1 antibodies elicited by this vaccine were cloned using peripheral blood B cells and sera collected two weeks after a one-year booster. Twelve of these fully human monoclonal antibodies (mAb) were tested for binding to MUC1+ target cells, and three with the highest binding were further evaluated for various effector functions important for tumor rejection. METHODS: Immune cells were incubated together with target cells expressing variations in the number, distance, and membrane anchoring properties of the MUC1 epitope in the presence of each mAb. RESULTS: All three mAbs mediated antibody-dependent cytokine release (ADCR), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Two also mediated antibody-dependent trogocytosis/trogoptosis (ADCT). None were capable of complement-dependent cytotoxicity (CDC). CONCLUSIONS: ADCP and ADCT functions were more efficient when antibodies bound epitopes proximal to and anchored to the membrane, providing insight for future therapeutic antibody validation strategies.

Synthesis and Thermodynamic Evaluation of Sialyl-Tn MUC1 Glycopeptides Binding to Macrophage Galactose-Type Lectin.

Interactions between the tumor-associated carbohydrate antigens of Mucin 1 (MUC1) and the carbohydrate-binding proteins, lectins, often lead to the creation of a pro-tumor microenvironment favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells that facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of their binding interaction is essential for it to be exploited for cancer vaccine strategies. The synthesis of MUC1 glycopeptide models carrying a single or multiple Tn and/or sialyl-Tn antigen(s) is described. A novel approach for the sialyl-Tn threonine building block suitable for the solid phase peptide synthesis was developed. The thermodynamic profile of the binding interaction between the human MGL and MUC1 glycopeptide models was analyzed using isothermal titration calorimetry. The measured dissociation constants for the sialyl-Tn-bearing peptide epitopes were consistently lower compared to the Tn antigen and ranged from 10 µM for mono- to 1 µM for triglycosylated MUC1 peptide, respectively. All studied interactions, regardless of the glycan's site of attachment or density, exhibited enthalpy-driven thermodynamics.

Amadori and Heyns rearrangement products of bioactive peptides as potential new ligands of galectin-3.

Non-enzymatic cascade reactions between amines and reducing sugars are known as Maillard reaction. The late phase of these reactions consists of advanced glycation end products (AGEs), which have been implicated in the pathogenesis of numerous human diseases. Recent evidence suggests that galectin-3 acts as a receptor for AGEs and some early products of the Maillard reaction. The early phase of the Maillard reaction, which consists of 1-amino-1-deoxyketoses (Amadori compounds) and 2-amino-2-deoxyaldoses (Heyns compounds), was the subject of our study. The binding interactions between galectin-3 and the Amadori and Heyns compounds of leucine-enkephalin (YGGFL), leucine-enkephalin methyl ester (YGGFL-OMe), truncated enkephalin (YGG and Y) and tetrapeptide (LSKL) were measured using the AlphaScreen competitive binding assay. The affinity of galectin-3 for Amadori and Heyns compounds depends on both the sugar moiety and the amino acid sequence of the model compounds. The best results were obtained with Leu-enkephalin derivatives of Amadori (IC50 = 6.06 µm) and Heyns (IC50 = 8.6 µm) compound, respectively.

Targeting Siglec-Sialylated MUC1 Immune Axis in Cancer.

Siglecs play a key role in mediating cell-cell interactions via the recognition of different sialylated glycoconjugates, including tumor-associated MUC1, which can lead to the activation or inhibition of the immune response. The activation occurs through the signaling of Siglecs with the cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins, while the inhibition signal is a result of the interaction of intracellular immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptors. The interaction of tumor-associated MUC1 sialylated glycans with Siglecs via ITIM motifs decreases antitumor immunity. Consequently, these interactions are expected to play a key role in tumor evasion. Efforts to modulate the response of immune cells by blocking the immune-suppressive effects of inhibitory Siglecs, driving immune-activating Siglecs, and/or altering the synthesis and expression of the sialic acid glycocalyx are new therapeutic strategies deserving further investigation. We will highlight the role of Siglec's family receptors in immune evasion through interactions with glycan ligands in their natural context, presented on the protein such as MUC1, factors affecting their fine binding specificities, such as the role of multivalency either at the ligand or receptor side, their spatial organization, and finally the current and future therapeutic interventions targeting the Siglec-sialylated MUC1 immune axis in cancer.

Exploring Glycan Binding Specificity of Odorranalectin by Alanine Scanning Library.

Fluorescently labelled alanine scan analogues of odorranalectin (OL), a cyclic peptide that exhibits lectin like properties, were screened for binding BSA-conjugated monosaccharides using an enzyme-linked lectin assay (ELLA). Results revealed that Lys5, Phe7, Tyr9, Gly12, Leu14, and Thr17 were crucial for binding BSA-L-fucose, BSA-D-galactose and BSA-N-acetyl-D-galactosamine. Notably, Ala substitution of Ser3, Pro4, and Val13 resulted in higher binding affinities compared to the native OL. The obtained data also indicated that Arg8 plays an important role in differentiation of binding for BSA-L-fucose/D-galactose from BSA-N-acetyl-D-galactosamine. The thermodynamics of binding of the selected alanine analogues was evaluated by isothermal titration calorimetry. Low to moderate binding affinities were determined for the tetravalent MUC1 glycopeptide and asialofetuin, respectively, and high for the fucose rich polysaccharide, fucoidan. The thermodynamic profile of interactions with asialofetuin exhibits shift to an entropy-driven mechanism compared to the fucoidan, which displayed an enthalpyentropy compensation, typically associated with the carbohydratelectin recognition process.

Multicomponent reaction derived small di- and tri-carbohydrate-based glycomimetics as tools for probing lectin specificity.

Lectins, carbohydrate-binding proteins, play important functions in all forms of life from bacteria and viruses to plants, animals, and humans, participating in cell-cell communication and pathogen binding. In an attempt to modify lectin functions, artificial lectin ligands were made usually as big dendrimeric or cluster multivalent glycomimetic structures. Here we synthesized a novel set of glycomimetic ligands through protection/deprotection multicomponent reactions (MCR) approach. Multivalent di-and tri-carbohydrate glycomimetics containing D-fructose, D-galactose, and D-allose moieties were prepared in 63-96% yield. MCR glycomimetics demonstrated different binding abilities for plant lectins Con A and UEA I, and human galectin-3. Information gained about the influence of molecule structure, multivalency and optical purity on the lectin binding ability can be used in lectin detection and sensitivity measurements to further facilitate understanding of carbohydrate recognition process.

Mucin-Type O-Glycosylation Proximal to ß-Secretase Cleavage Site Affects APP Processing and Aggregation Fate.

The amyloid-ß precursor protein (APP) undergoes proteolysis by ß- and γ-secretases to form amyloid-ß peptides (Aß), which is a hallmark of Alzheimer's disease (AD). Recent findings suggest a possible role of O-glycosylation on APP's proteolytic processing and subsequent fate for AD-related pathology. We have previously reported that Tyr681-O-glycosylation and the Swedish mutation accelerate cleavage of APP model glycopeptides by ß-secretase (amyloidogenic pathway) more than α-secretase (non-amyloidogenic pathway). Therefore, to further our studies, we have synthesized additional native and Swedish-mutated (glyco)peptides with O-GalNAc moiety on Thr663 and/or Ser667 to explore the role of glycosylation on conformation, secretase activity, and aggregation kinetics of Aß40. Our results show that conformation is strongly dependent on external conditions such as buffer ions and solvent polarity as well as internal modifications of (glyco)peptides such as length, O-glycosylation, and Swedish mutation. Furthermore, the level of ß-secretase activity significantly increases for the glycopeptides containing the Swedish mutation compared to their nonglycosylated and native counterparts. Lastly, the glycopeptides impact the kinetics of Aß40 aggregation by significantly increasing the lag phase and delaying aggregation onset, however, this effect is less pronounced for its Swedish-mutated counterparts. In conclusion, our results confirm that the Swedish mutation and/or O-glycosylation can render APP model glycopeptides more susceptible to cleavage by ß-secretase. In addition, this study sheds new light on the possible role of glycosylation and/or glycan density on the rate of Aß40 aggregation.

Exploring the Galectin Network by Light and Fluorescence Microscopy.

Dynamic changes of a cell's glycophenotype are increasingly interpreted as shifts in the capacity to interact with tissue (endogenous) lectins. The status of glycan branching or chain length (e.g., core 1 vs core 2 mucin-type O-glycans and polyLacNAc additions) as well as of sialylation/sulfation has been delineated to convey signals. They are "read" by galectins, for example regulating lattice formation on the membrane and cell growth. Owing to the discovery of the possibility that these effectors act in networks physiologically resulting in functional antagonism or cooperation, their detection and distribution profiling need to be expanded from an individual (single) protein to the-at best-entire family. How to work with non-cross-reactive antibodies and with the labeled tissue-derived proteins (used as probes) is exemplarily documented for chicken and human galectins including typical activity and specificity controls. This description intends to inspire the systematic (network) study of members of a lectin family and also the application of tissue proteins beyond a single lectin category in lectin histochemistry.

What is the Sugar Code?

A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e. g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysaccharides are ubiquitous, and they are a biochemical platform for establishing molecular messages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding capacity by making an unsurpassed versatility for isomer (code word) formation possible by variability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins 'read' the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/π-interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar code.

Tyrosine O-GalNAc Alters the Conformation and Proteolytic Susceptibility of APP Model Glycopeptides.

The amyloid-ß precursor protein (APP) undergoes proteolytic cleavage by α-, ß-, and γ-secretases, to determine its fate in Alzheimer's disease (AD) pathogenesis. Recent findings suggest a possible role of O-glycosylation in APP's proteolytic processing. Therefore, we synthesized native and Swedish-double-mutated APP (glyco)peptides with Tyr681-O-GalNAc. We studied conformational changes and proteolytic processing using circular dichroism (CD) spectroscopy and enzyme cleavage assay, respectively. CD analysis was carried out in four solvent systems to evaluate peptide environment and O-glycosylation induced conformational changes. The Swedish mutation and Tyr681-O-GalNAc were the key factors driving conformational changes. Furthermore, the level of α- and ß-secretase activity was increased by the presence of mutation and this effect was more pronounced for its glycosylated analogues. Our results suggest that O-glycosylation of Tyr681 can induce a conformational change in APP and affect its proteolytic processing fate toward the amyloidogenic pathway.

Imitating evolution's tinkering by protein engineering reveals extension of human galectin-7 activity.

Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.

Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin.

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.

Galectin-Glycan Interactions: Guidelines for Monitoring by 77 Se NMR Spectroscopy, and Solvent (H2 O/D2 O) Impact on Binding.

Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance. Their study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as S- and Se-glycosides. The latter enable extensive analyses by specific 77 Se NMR spectroscopy, but still remain underexplored. By using the example of selenodigalactoside (SeDG) and the human galectin-1 and -3, we have evaluated diverse 77 Se NMR detection methods and propose selective 1 H,77 Se heteronuclear Hartmann-Hahn transfer for efficient use in competitive NMR screening against a selenoglycoside spy ligand. By fluorescence anisotropy, circular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDG binding by galectins are similar to thiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact. ITC data in D2 O versus H2 O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAc dimers with extended chain length.

Modulation of receptor binding to collagen by glycosylated 5-hydroxylysine: Chemical biology approaches made feasible by Carpino's Fmoc group.

The creation of the 9-fluorenylmethoxycarbonyl (Fmoc) group by the Carpino laboratory facilitated the synthesis of peptides containing acid-sensitive groups, such as O-linked glycosides. To fully investigative collagen biochemistry, one needs to assemble peptides that possess glycosylated 5-hydroxylysine (Hyl). A convenient method for the synthesis of Fmoc-Hyl(ε-tert-butyloxycarbonyl (Boc),O-tert-butyldimethylsilyl (TBDMS)) and efficient methods for the synthesis of Fmoc-Hyl[ε-Boc,O-(2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl)] have been developed. Glycosylated Fmoc-Hyl derivatives were used to construct a series of types I-IV collagen-model triple-helical peptides (THPs) that incorporated known or proposed receptor binding sites. Glycosylation of Hyl was found to strongly down-regulate the binding of CD44 and the α3ß1 integrin to collagen, while the impact on α2ß1 integrin binding was more modest. Molecular modeling of integrin binding indicated that Hyl glycosylation directly impacted the association between the α3ß1 integrin metal ion-dependent adhesion site (MIDAS) and the receptor binding site within type IV collagen. The Fmoc solid-phase strategy ultimately allowed for chemical biology approaches to be utilized to study tumor cell interactions with glycosylated collagen sequences and document the modulation of receptor interactions by Hyl posttranslational modification.

TF-containing MUC1 glycopeptides fail to entice Galectin-1 recognition of tumor-associated Thomsen-Freidenreich (TF) antigen (CD176) in solution.

Aberrant Mucin-1 (MUC1) glycosylation with the Thomsen-Friedenreich (TF) tumor-associated antigen (CD176) is a hallmark of epithelial carcinoma progression and poor patient prognosis. Recognition of TF by glycan-binding proteins, such as galectins, enables the pathological repercussions of this glycan presentation, yet the underlying binding specificities of different members of the galectin family is a matter of continual investigation. While Galectin-3 (Gal-3) recognition of TF has been well-documented at both the cellular and molecular level, Galectin-1 (Gal-1) recognition of TF has only truly been alluded to in cell-based platforms. Immunohistochemical analyses have purported Gal-1 binding to TF on MUC1 at the cell surface, however binding at the molecular level was inconclusive. We hypothesize that glycan scaffold (MUC1's tandem repeat peptide sequence) and/or multivalency play a role in the binding recognition of TF antigen by Gal-1. In this study we have developed a method for large-scale expression of Gal-1 and its histidine-tagged analog for use in binding studies by isothermal titration calorimetry (ITC) and development of an analytical method based on AlphaScreen technology to screen for Gal-1 inhibitors. Surprisingly, neither glycan scaffold or multivalent presentation of TF antigen on the scaffold was able to entice Gal-1 recognition to the level of affinity expected for functional significance. Future evaluations of the Gal-1/TF binding interaction in order to draw connections between immunohistochemical data and analytical measurements are warranted.

Correction to: Influence of protein (human galectin-3) design on aspects of lectin activity.

In the original publication of the article, the "Result" section has been published.

Influence of protein (human galectin-3) design on aspects of lectin activity.

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure-activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.

Tumor-associated O-glycans of MUC1: Carriers of the glyco-code and targets for cancer vaccine design.

The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. It has frequently been reported that MUC1, the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern, in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play a key role in tumor initiation, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with tumor escape from immune defenses. In this report, we will give an overview of the oncogenic functions of MUC1 that are exerted through TACA interactions with endogenous carbohydrate-binding proteins (lectins). These interactions often lead to creation of a pro-tumor microenvironment, favoring tumor progression and metastasis, and tumor evasion. In addition, we will describe current efforts in the design of cancer vaccines with special emphasis on synthetic MUC1 glycopeptide vaccines. Analysis of the key factors that govern structure-based design of immunogenic MUC1 glycopeptide epitopes are described. The role of TACA type, position, and density on observed humoral and cellular immune responses is evaluated.

Positional Scanning MUC1 Glycopeptide Library Reveals the Importance of PDTR Epitope Glycosylation for Lectin Binding.

One of the main barriers to explaining the functional significance of glycan-based changes in cancer is the natural epitope heterogeneity found on the surface of cancer cells. To help address this knowledge gap, we focused on designing synthetic tools to explore the role of tumor-associated glycans of MUC1 in the formation of metastasis via association with lectins. In this study, we have synthesized for the first time a MUC1-derived positional scanning synthetic glycopeptide combinatorial library (PS-SGCL) that vary in number and location of cancer-associated Tn antigen using the "tea bag" approach. The determination of the isokinetic ratios necessary for the equimolar incorporation of (glyco)amino acids mixtures to resin-bound amino acid was determined, along with developing an efficient protocol for on resin deprotection of O-acetyl groups. Enzyme-linked lectin assay was used to screen PS-SGCL against two plant lectins, Glycine max soybean agglutinin and Vicia villosa. The results revealed a carbohydrate density-dependent affinity trend and site-specific glycosylation requirements for high affinity binding to these lectins. Hence, PS-SGCLs provide a platform to systematically elucidate MUC1-lectin binding specificities, which in the long term may provide a rational design for novel inhibitors of MUC1-lectin interactions involved in tumor spread and glycopeptide-based cancer vaccines.

How altering the modular architecture affects aspects of lectin activity: case study on human galectin-1.

Discoveries on involvement of glycan-protein recognition in many (patho)physiological processes are directing attention to exploring the significance of a fundamental structural aspect of sugar receptors beyond glycan specificity, i.e., occurrence of distinct types of modular architecture. In order to trace clues for defining design-functionality relationships in human lectins, a lectin's structural unit has been used as source material for engineering custom-made variants of the wild-type protein. Their availability facilitates comparative analysis toward the stated aim. With adhesion/growth-regulatory human galectin-1 as example, the strategy of evaluating how changes of its design (here, from the homodimer of non-covalently associated domains to (i) linker-connected di- and tetramers and (ii) a galectin-3-like protein) affect activity is illustrated by using three assay systems of increasing degree of glycan complexity. Whereas calorimetry with two cognate disaccharides and array testing with 647 (glyco)compounds disclosed no major changes, galectin histochemical staining profiles of tissue sections that present natural glycome complexity revealed differences between wild-type and linker-connected homo-oligomers as well as between the galectin-3-like variant and wild-type galectin-3 for cell-type positivity, level of intensity at the same site and susceptibility for inhibition by a bivalent glycocompound. These results underscore the strength of the documented approach. Moreover, they give direction to proceed to (i) extending its application to other members of this lectin family, especially galectin-3 and (ii) then analyzing impact of architectural alterations on cell surface lattice formation and ensuing biosignaling systematically, considering the variants' potential for translational medicine.